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Keywords:

  • Hodgkin lymphoma;
  • IRF4/MUM1;
  • CD40;
  • CD95/FAS;
  • chemotherapy

Summary

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results and discussion
  5. Acknowledgements
  6. References

The effects of proliferative, apoptotic and anti-proliferative stimuli on interferon regulatory factor 4 (IRF4) expression by Reed-Sternberg (RS) cells were analysed using a panel of Hodgkin lymphoma (HL)-derived cell lines. IRF4 expressed by HL cells was consistently upregulated after CD40 engagement; IRF4 was downregulated by agonistic anti-CD95 antibodies in the FAS-sensitive HDLM-2 cells and after treatment with Adriamycin and Dacarbazine, two chemotherapic agents commonly used for HL treatment. These results demonstrated, for the first time, that IRF4 was up-modulated by CD40 engagement, and down-modulated by apoptotic and anti-proliferative signals, suggesting an involvement of IRF4 also in HL pathobiology.

Interferon Regulatory Factor 4 (IRF4), a member of the IRF family of transcription factors, is a molecule required for lymphocyte activation and generation of immunoglobulin-secreting plasma cells during immune responses (Klein et al, 2006) and represents a key regulator of several steps in myeloid- and dendritic-cells differentiation (Tamura et al, 2005). IRF4 plays an essential role in multiple myeloma proliferation and cell survival and in Epstein–Bar virus (EBV)-mediated transformation of human B lymphocytes (Shaffer et al, 2009; Xu et al, 2008). Although several reports have demonstrated that IRF4 is also expressed in Reed-Sternberg (RS) cells of classical-Hodgkin Lymphoma (cHL) (Carbone et al, 2001, 2002; Falini et al, 2000; Valsami et al, 2007), its possible role in cHL cell growth and survival has not yet been investigated.

Deregulation of multiple signalling pathways and of downstream transcription factors, including Nuclear Factor-kappa B (NF-κB), is a further hallmark of RS cells (Küppers, 2009). CD40 activation leads to NF-κB mediated induction of IRF4 in normal B-cells (Saito et al, 2007). IRF4 and CD40 are consistently expressed by RS cells of primary cHL. CD40 signalling is involved in RS cell survival and proliferation (Carbone et al, 1995; Küppers, 2009) and we speculated whether CD40L, a survival signal provided by rosetting T cells of the HL microenvironment (Carbone et al, 1995; Küppers, 2009), might affect IRF4 expression. This study demonstrated that IRF4, as expressed by HL cells, was up-modulated by CD40 engagement and down-modulated by apoptotic and anti-proliferative signals.

Materials and methods

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results and discussion
  5. Acknowledgements
  6. References

Cell lines and T cells purification

The study utilized a set of human HL-derived cell lines: KM-H2, HDLM-2 and L-428 were obtained through the German collection of Microorganisms and cell cultures (Braunschweig, Germany), L-1236 and L540 cell lines were kindly provided by A. Jox and V. Diehl (University of Koln, Germany) respectively. The myeloid leukaemia cell line HEL (negative control for IRF4) and the multiple myeloma cell line IM-9 (positive control for IRF4) were obtained from the American Type Culture collection (Rockville, MD, USA). The mouse fibroblastic Ltk-cell line stably transfected with CD40L and the non-transfected L cell line were kindly provided by Dr P. De Paoli (CRO, Aviano). CD4+ T cells were isolated from the peripheral blood mononuclear cell fraction using a cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. All cell lines were maintained in Iscove’s modified Dulbecco’s medium (IMDM) (Biocrom KG, Berlin, Germany) supplemented with 10% heat inactivated foetal calf serum (FCS; Sigma-Aldrich, Milan, Italy), 0·2 mg/ml penicillin/streptomycin and 0·1% (w/v) l-glutamine (Biocrom) at 37°C in a 5% CO2 fully humidified atmosphere. CD4+ T cells were activated with tetradecanoylphorbol-13-acetate (TPA; Sigma-Aldrich) (10 ng/ml) and ionomycin (Sigma-Aldrich) (1 μg/ml).

Flow cytometry, western blotting and immunocytochemistry.

Interferon Regulatory Factor 4 expression was analysed by indirect immunofluorescence, Western blot analysis and immunocytochemistry as previously reported (Aldinucci et al, 2008; Carbone et al, 2002) using an affinity-purified polyclonal goat antibody (M-17) specific for the IRF4 protein (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).

Annexin-V binding was detected by flow cytometry with fluorescein isothiocyanate (FITC)-conjugated Annexin-V protein (BD Pharmingen, Immunocytometry System, San Jose, CA, USA).

Modulation of IRF4 expression

After an overnight incubation in serum-free (SF) medium, 5 × 105/ml HL cells were cultured in IMDM with 1% FCS in the presence of sCD40L (1 μg/ml) and the enhancer (1 μg/ml) (Aldinucci et al, 2008). Alternatively, CD40+ HL cells (1·5 × 105/ml) were cultured in the presence of 0·5 × 106/well mitomycin-C treated (Sigma) (50 μg/ml, 2·5 h at 37°C) non-transfected or transfected with human CD40-ligand (HuCD40L-Lcells) murine Ltk-cells (Aldinucci et al, 2008) in SF medium. CD95-mediated apoptosis was induced by incubating HDLM-2 and L-1236 cells (5·0 × 104/ml) in IMDM containing 5% FCS for 24 h in the presence of 100 ng/ml of the agonistic anti-CD95 monoclonal antibody (mAb) CH-11 (Medical & Biological Laboratories, Nagoya, Japan) or an isotype-matched control antibody. In other experiments HL-cell lines (2 × 105/ml) were incubated with increasing concentrations of Adriamycin (ADM) (Pharmacia & Upjohn, Milan, Italy) or Dacarbazine (DTIC) (Aventis, Milan, Italy). After 72 h, viable cells were counted, recovered and protein content and apoptosis analysed. IRF4 expression was analysed by Western blotting, always using equivalent amounts of protein or by flow cytometry and identified by Alexa fluor 488 donkey anti Goat IgG (Invitrogen, Milan, Italy).

Results and discussion

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results and discussion
  5. Acknowledgements
  6. References

Cell lines represent the only suitable model in HL to perform functional studies, Thus, IRF4 expression on a panel of HL-derived cell lines, was first investigated. Strong IRF4 expression was detected by flow cytometry (Fig 1A), Western blotting (Fig 1B) and immunocytochemistry (Fig 1C) in all HL cell lines tested, excluding L540 cells. Immunoreactive IRF4 migrated as a single component of about 50 kDa identical to that expressed by IM9 cells (positive control) (Falini et al, 2000).

image

Figure 1.  Expression of IRF4 on HL-derived cell lines. (A) HL cells and activated T cells (positive control) were stained with anti-IRF4 antibodies. Thin lines indicate background fluorescence, as determined by isotype-matched control immunoglobulins. The X- and Y-axes indicate the logarithm of the relative intensity of red fluorescence and relative cell number respectively. (B) Proteins (7 μg/lane) from HL cells, HEL cells (negative control) and IM-9 cells (positive control), were subjected to 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. Blots were then incubated with anti-human anti-IRF4 antibodies and developed by chemiluminescence. (C) Immunostaining patterns of HL cell lines with anti-IRF4 antibodies. Cell blocks, immunoperoxidase, haematoxylin counterstain. Images were taken using a Nikon Eclipse 80i microscope (Nikon, Tokyo, Japan) with a pan fluor 40 × /0·75 objective and Nikon digital sight DS-Fi1 camera equipped with control unit-DS-L2 (Nikon) (original magnification × 30).

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Interferon Regulatory Factor 4 expression was evaluated in the presence of anti-apoptotic or anti-proliferative signals. CD40, a molecule expressed by RS cells (Carbone et al, 1995), is involved in HL cells proliferation and survival, and protects HDLM-2 cells from FAS-induced apoptosis (Skinnider & Mak, 2002). CD40 engagement on normal B cells activates NF-κB, which in turn induces IRF4 expression (Saito et al, 2007). Thus, we assumed that RS cells of HL may also receive signals via the CD40 receptor (Carbone et al, 1995; Küppers, 2009; Skinnider & Mak, 2002) and modulate IRF4 expression. Consistent with this, CD40 engagement by both soluble- and membrane-bound-CD40L resulted in an upregulation of IRF4 in all HL cell lines tested (Fig 2A). This finding suggests that the high levels of IRF4 expressed by RS cells may be associated with CD40 engagement, and reinforces the notion that CD40L+ rosetting T cells of the HL microenvironment may contribute to the pathogenesis of HL. EBV is often found in HL (Küppers, 2009). Given that Latent Membrane Protein 1 (LMP1), a protein encoded by EBV, appears to mimic CD40 in multiple ways, we could not exclude that IRF4 expression by RS cells in vivo might be related, at least in part, to EBV infection (Xu et al, 2008).

image

Figure 2.  IRF4 expression in HL-derived cell lines is modulated by CD40L, CD95 engagement and drug treatment. (A) After an overnight incubation in serum-free medium, 5 × 105/ml HL cells were cultured in IMDM with 1% FCS in the presence of sCD40L (1 μg/ml) and the enhancer (1 μg/ml)(upper panel). Alternatively, 1·5 × 105/ml HL cells were cultured in the presence of non-transfected or human CD40-ligand transfected murine Ltk-cells in serum-free medium (lower panel). After 72 h, proteins (7 μg/lane) were analysed for IRF4 by western blotting assay. (B) HDLM-2 and L-1236 cells (5·0 × 104/ml) were cultured in IMDM containing 5% FCS for 24 h in the presence of 100 ng/ml of the agonistic anti-CD95 mAb CH-11. Proteins (7 μg/lane) were analysed for IRF4 by western blotting assay. (C) HL-cell lines (2 × 105/ml) were incubated with increasing amounts of ADM and DTIC. After 72 h cells were stained with the vital dye trypan blue and the number of viable cells counted. Results are the mean values of three independent experiments and errors are shown as the standard deviation. (D) HL-cell lines (2 × 105/ml) were cultured with ADM (0·5 μmol/l) or DTIC (800 μg/ml). After 72 h cells were recovered and proteins (7 μg/lane) analysed for IRF4 by western blotting assay (E) Fluorescence histograms showing apoptosis induction and IRF4 decrease after ADM and DTIC treatment in both L-1236 and L-428 cells. Apoptosis was evaluated by staining with Annexin-V-FITC (top). Dotted lines represent Annexin-V binding in the absence of drugs. IRF4 expression was analysed with anti-IRF4 antibodies (bottom) followed by Alexa fluor 488 donkey anti-goat and analysed by flow cytometry. Dotted lines indicate background fluorescence. The X- and Y-axes indicate the logarithm of the relative intensity of green fluorescence and relative cell number respectively.

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Interferon Regulatory Factor 4 is downregulated by apoptotic and anti-proliferative stimuli in multiple myeloma (Verdelli et al, 2009). To assess the effects of CD95-mediated apoptosis on IRF4 expression in HL, we used the CD95L-sensitive HDLM-2 cells and the CD95L-resistant L-1236 cells (Re et al, 2000). The agonistic anti-CD95 CH11 mAb, together with a strong apoptotis induction (data not shown), remarkably decreased IRF4 expression in HDLM-2 cells, as evaluated by both Western blotting and flow cytometry (data not shown). As expected, L-1236 cells were unaffected, (Fig 2B), suggesting that the effects of CD95 ligation are specific.

We evaluated IRF4 expression and apoptosis after treatment with ADM and DTIC, two chemotherapeutic agents commonly used for HL treatment. HL-cell lines were incubated with increasing concentrations of ADM and DTIC and IRF4 expression and apoptosis were evaluated using a drug concentration (0·5 μmol/l for ADM and 800 μg/ml for DTIC) capable of inducing about 75% of growth inhibition in all HL cell lines tested. As shown in Fig 2C both drugs produced a dose-response inhibition of HL proliferation, with KM-H2 cells being the most sensitive. Both ADM and DTIC determined a remarkable decrease of IRF4 expression in all HL cell lines tested (Fig 2D), as evaluated by western Blot. ADM (0·5 μmol/l) and DTIC (800 μg/ml), induced apoptosis in both L-1236 and L-428 cells, as evaluated by Annexin-V binding (Fig 2E, top), together with the downregulation of IRF4 expression, as evaluated, in this case, by flow cytometry (Fig 2E, bottom).

IRF4 downregulation inhibited the proliferation of both EBV-transformed B-cells and multiple myeloma cells, and the present data raise the possibility that IRF4 may also be involved in RS cells proliferation.

Acknowledgements

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results and discussion
  5. Acknowledgements
  6. References

Supported in part by the Associazione Italiana per la Ricerca sul Cancro (A.I.R.C.), Milan, Italy and the Ministero della Salute, Ricerca Finalizzata FSN, I.R.C.C.S., Rome, Italy (to D.A.); and by a grant from the Ministero della Salute, Rome, within the framework of the Progetto Integrato Oncologia-Advanced Molecular Diagnostics ‘Multidimensional characterization of tumours’ project and the Ministero della Salute, Ricerca Finalizzata FSN, I.R.C.C.S., Rome, Italy (to A.Carbone). We thank Ms Cinzia Borghese for her help with the apoptosis analysis.

References

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results and discussion
  5. Acknowledgements
  6. References
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